Dr Michael Caves, India Business Development Manager, Malvern Instruments, gives an insight about orthogonal techniques from Malvern Instruments that can be used to quantify particles in this critically important size range in order to obtain insights that can speed up biopharmaceutical development and meet regulatory requirements
Particles over the range of 0.1µm to 10 µm are of particular interest in biopharmaceutical formulation for parenteral administered biologics, due to side effects such as loss of efficacy and autoimmunity that compromise drug safety and efficacy.
Submicron particles are particularly difficult to characterise. It is therefore essential to monitor aggregation in the development, manufacture and storage of therapeutic proteins. Subvisible aggregates are coming under increasing regulatory scrutiny, but are often inaccessible to optical methods due to limits on resolution.
Solving the problem
Resonant mass measurement – Differentiating protein particles from silicon
Archimedes is a highly innovative instrument which uses the technique of resonant mass measurement to detect and accurately count particles in the size range 50nm – 5µm, and reliably measure their buoyant mass, density, dry mass and size. Particularly useful for the characterisation of protein aggregates in formulation or buffer, Archimedes is also able to distinguish between proteinaceous material and contaminants such as silicone oil by means of comparing their relative resonant frequencies and buoyant masses. The technique works by passing the sample through a microfluidic channel embedded within a resonating cantilever (Figure 1). The frequency of cantilever resonance shifts either up or down as particles with densities that are different from that of the carrier solution flow through it. By precisely measuring the magnitude in excursion from the cantilever’s base line frequency, an accurate measurement of particle size can be derived for the particles within solution. The shear force exerted on a biopharmaceutical formulation during syringing has been identified as having the potential to cause aggregation. Quantifying the presence of aggregate species within formulation, pre, and post-syringing is, therefore, essential for monitoring the safety of administration.
The following case studies illustrate how this innovative analytical setup enables subvisible particle characterisation in bioformulation QC and process studies. (Figure 2) compares the number of subvisible particles within a control sample (blue) with those in a sample that has undergone syringe-induced shear (green). Prior to syringe stress, the number of particles detected is low demonstrating that the sample is reasonably pure and contains only a few large aggregates. This number increases significantly following the application of shear stress. It can be used to compare different stress conditions to provide an overall picture of the degradation profile for the biopharmaceutical of interest.
Nanoparticle tracking analysis
The Malvern NanoSight range (Figure 3) utilises Nanoparticle Tracking Analysis (NTA) to characterise particles from 10 nm – 2000 nm. Each particle is individually but simultaneously analysed by direct observation and measurement of diffusion events. Both particle size and concentration are measured, while an additional feature of fluorescence mode provides differentiation of labelled or naturally fluorescing particles. This particle-by-particle methodology produces high resolution size distribution profiles, whilst visualisation gives additional confidence for process optimisation. The ability of NTA to characterise sub-micron particles has attracted the attention of numerous workers in this field and the technique has been assessed and applied to the real-time study of proteinaceous aggregates and their formation in several applications.
To illustrate the role of NTA in subvisible particle characterisation, virus particles were quantified before and after being subjected to shaking-induced shear stress. (Figure 4a) shows a monodisperse peak with size 45 nm prior to stress. However, following agitation by simple shaking for a few seconds, shear stress was seen to have induced aggregation in the virus sample (Figure 4b). To prevent the presence of large aggregates rendering a protein therapeutic unsuitable for patients, an understanding of where in the process of synthesis, purification, packaging, transport, storage and use the proteins monomer units begin to aggregate together is required. Size distribution measurements with NTA at different points in the process enables identification of aggregation point that can then be reviewed and potentially modified to prevent or slow the formation of protein aggregates.
G3 – ID
The Morphologi G3-ID (Figure 5) offers a unique capability, combining the automated static imaging features of a hybrid optical microscope with chemical identification of individual particles using Morphologically-Directed Raman Spectroscopy to enable the measurement of component-specific particle size and shape distributions in the range of 1 µm to 1000 µm. Particulate enumeration and characterisation are of particular interest in the field of biotherapeutic formulation. Automated optical microscopes, such as the Morphologi G3-ID, are well-suited to operation in subvisible size range, and offer high-resolution images and 100 per cent particle counting for the sample volume delivered. Raman spectroscopy extends these capabilities, allowing for chemical identification of particles.
The case study explores the use of Morphologically-Directed Raman spectroscopy (MDRS) to identify and analyse contaminants including protein aggregates present in a stressed sample of lysozyme. The size groups defined for particle counting included 2 µm to 10 µm (small), 10 µm to 25 µm (medium), and greater than 25 µm (large), with the addition of a specific group for the 40 µm polystyrene latex spheres. A selection of images from each group of particles detected on the sample is shown in (Figure 6a). The polystyrene particles were bright white in colour due to the top-light (episcopic) illumination geometry used to collect data, while the aggregate appeared darker with a variety of shape characteristics as confirmed using Raman spectroscopy data (Figure 6b). Point Raman spectroscopy of individual particles allows identification of particles, such as glass, cellulose and silicon, commonly found in biopharmaceutical samples.
Figure 6. Representative (a) particle images from each size band defined for the lysozyme sample along with (b) Raman spectra collected from the lysozyme sample: cellulose (black), lysozyme aggregate (blue), polystyrene sphere (red). The inset (green) shows the spectrum of a rubber particle that was found in the sample
Malvern orthogonal approach
The complexities involved in bridging the subvisible gap for biotherapeutic development make an orthogonal approach to characterisation essential. This article outlines three of the many technologies that Malvern offers as a solution for the challenge of biopharmaceutical development, focussing on both large and small aggregates.
Together with our customers, we have created a suite of instruments which provide access to a raft of biophysical information on your product, to speed you through characterisation studies, from formulation right through to patient administration.
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